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Abstract

Background

High-throughput studies provide a wide spectrum of genes for use as predictive markers
during testicular sperm extraction (TESE) in combination with ICSI. In this work,
we used the specimens from testicular biopsies of men with non-obstructive azoospermia
who underwent TESE to investigate the expression of spermatogenesis-related genes
MND1, SPATA22, GAPDHS and ACR.

Methods

Testicular biopsy specimens were subdivided into three groups: hypospermatogenesis
(HS); maturation arrest (MA); and Sertoli cell-only syndrome (SCO). The levels of
expression of the spermatogenesis-related genes MND1, SPATA22, GAPDHS and ACR in the testes were compared among these three groups using the reverse transcription
polymerase chain reaction (RT-PCR) technique.

Results

Analysis of the expression of spermatogenic genes in human testes with abnormal spermatogenesis
showed different expression patterns in patients from different groups. Fertilization
rate for studied set of patients was 66% and pregnancy rate 29%. For HS group fertilization
rate was 72% and pregnancy rate 32%, while for MA group fertilization and pregnancy
rates were 54% and 26%, respectively. Fertilization rates in relation to the studied
genes were uniformly around 70%, pregnancy rates for ACR and GAPDHS genes were surprisingly
low at 6% and 8% correspondingly.

Conclusions

Analysis of the expression of genes involved in spermatogenesis can be a fast additional
test for the level of spermatogenesis in testicular samples.

Keywords:

Background

Testicular tissue is composed of many cell types serving as spatio-temporal environment
for the male germ cell development. It is the only place in the male organism where
meiosis occurs. After meiotic division, gene expression continues in haploid cells
until chromatin condensation to produce proteins necessary for the final stages of
spermatogenesis [1]. Germ cells also employ mechanisms for mRNA storage and delayed translation after
chromatin has already been packaged. The final products of spermiogenesis are highly
differentiated sperm cells, which are transcriptionally inactive. The rate of cell
proliferation in testicular tissue is higher than in other tissues due to continuous
sperm production. All these facts make gene expression analysis of testicular tissues
extremely important.

Changes in the complex process of spermatogenesis caused by genetic background or
environmental factors can lead to male infertility. Infertile men with no sperm cells
in the ejaculate can father a child with the help of assisted reproduction techniques
using testicular sperm. Intracytoplasmic sperm injection (ICSI) can be successful
in men with non-obstructive azoospermia, but it cannot help patients with Sertoli
cell-only (SCO) syndrome. Genome-wide expression studies of large groups of patients
were performed to analyse the general changes in global gene expression of patients
with infertility phenotypes [2-5]. This led to the identification of gene clusters that were differentially expressed
in patients with spermatogenesis defects [4]. High-throughput studies provide a wide spectrum of genes for use as markers in the
combination of testicular sperm extraction (TESE) with ICSI. Forty-seven genes exhibiting
differential testicular gene expression associated with male infertility were detected
in mice and 19 in humans [6]. They included genes involved in DNA repair, glutathione metabolism, proteolysis,
spermatogenesis and stress response. These findings enabled the identification of
markers for specific stages of spermatogenesis and the presence of somatic cells,
thus improving infertility diagnostics.

In this work, we used the specimens from testicular biopsies of infertile men who
underwent TESE for the ICSI procedure and investigated the expression of spermatogenesis-related
genes. The GAPDH gene is expressed in somatic testicular cells and spermatogonia. Two genes, MND1/GAJ and SPATA22, are expressed prior to meiotic division and their corresponding proteins are involved
in meiotic progression during spermatogenesis. GAPDHS and ACR genes reach the highest expression levels in haploid spermatids and are important
for the sperm function.

Methods

Patients

Testicular tissue samples were collected from patients treated for infertility in
ISCARE I.V.F. a.s. A total of 47 biopsy samples were obtained from azoospermic men
aged 27–63 years. All patients enrolled in the study underwent testicular biopsies
within their treatment and gave their written informed consent with donating the used
material for the purposes of this research project. The study was approved by the
institutional review board at the Institute of Biotechnology.

TESE procedure, sperm extraction and ICSI

Testicular sperm extraction (TESE) was performed as previously described [7]. Briefly, small pieces of testicular tissue were placed in a Petri dish in Flushing
medium (Medicult, Copenhagen, Denmark) and cupped up using two sterile needles. The
fragmented tissue was assessed for the presence of motile spermatozoa under the phase
contrast microscope. The suspension of cells was cultivated for 24–48 hours before
injection or freezing procedure. Prior to the sperm retrieval procedure, a small piece
of testicular tissue was taken for histological examination.

TESE samples were divided into three groups: hypospermatogenesis (HS), maturation
arrest (MA), and Sertoli cells only syndrome (SCO), with a histopathology score counting
according to Holstein et al. [8]. The corresponding grades were 6–8 for HS, grades 3–5 for MA and grade 2 for the
SCO group.

ICSI procedures were carried out according to Silber et al. [9]. The sperm cells were incubated in droplets of 5 μl of Flushing medium (Medicult)
with 30% of human serum for 2 hours followed by injection into the oocyte. The fertilization
rate was assessed approximately 18 h after the injection by the presence of two pronuclei
and second polar body and was quantified as a percentage of fertilized mature oocytes.
Clinical pregnancy was confirmed by observing the gestational sac or detecting foetal
heart beats.

RNA purification

RNA purification was performed with the same piece of testicular tissue that was used
for the sperm extraction and subsequently cryopreserved. Testicular biopsies with
the residual medium were thawed directly in RNAlater RNA Stabilization Reagent (Qiagen,
Chatsworth CA) and samples were homogenized with the Precellys 24 tissue homogenizer
(Bertin Technologies, France). Total RNA was purified from the tissue samples using
the RNeasy lipid tissue mini kit (Qiagen, Chatsworth CA) according to the manufacturer’s
instructions and stored at −70°C. The concentration and purity of the purified RNA
was determined by UV spectrophotometer Helios α (Thermo Electron Corporation, Marietta,
USA) and confirmed by agarose gel electrophoresis. Human total testicle RNA, 1 mg/ml
(Ambion®, Life Technologies™, Carlsbad CA), was used as a positive control.

RT-qPCR

Reverse transcription and subsequent RT-qPCR was performed as previously described
[10]. Briefly, prior to reverse transcription, purified RNA was treated with RNAse-free
DNAse 1 (Fermentas, Burlington, Canada) for 40 min. Template cDNA was synthesized
from 1 μg of total testicular RNA using SuperScript® III Reverse Transcriptase (Invitrogen,
Life Technologies, Carlsbad CA) or RevertAid™ Reverse Transcriptase (Fermentas, Burlington,
Canada) with combination of random hexamer and poly(dT) primers (1:1) in a Touchgene
Gradient Thermal Cycler (Techne, Burlington, USA). The qPCR conditions were: initial
denaturation for 15 min, followed by 40 cycles of denaturation at 94°C for 20 sec,
annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. Following PCR reaction,
the melting curve was constructed by increasing the temperature from 72 to 95°C to
ensure that the correct product is amplified in the reaction. PCR was repeated three
times in doublets for each gene, and the average Ct was used for further analysis.
Gene-specific primers for PPIA, GAPDHS and ACR were designed using the advantages of Primer3 software and BLAST alignment of Primer-BLAST
service from NCBI [11]. Three best primer pairs overlapping the intron sequence were ordered and after pretesting,
the best of them was used for gene expression analysis. Due to the large amount of
pseudogenes for GAPDH gene in human genome [12], neither of the three primer pairs ordered was suitable because of the dimer formations,
and the primers used were as in Barber et al. [13]. Primers for MND1 and SPATA22 were as in Okada et al. [4]. The PPIA (peptidylprolyl isomerase A (cyclophilin A)) gene was used as a reference gene. Primer
properties are summarized in Table 1.

Statistical analysis

Experimental data were analysed using STATISTICA 6.0. and GraphPad Prism 5.04. The
differences between the control and experimental groups in the relative gene expression
were analysed by KW ANOVA, and post hoc analysis was performed by Dunn‘s test. The
p value that was equal to or lower than 0.05 was considered to be significant and
was indicated with red asterisk in the column.

Results

A total of 47 testicular biopsies were analysed. As the specimens were primarily used
for sperm retrieval, in 13 cases the level or purity of isolated RNA was not sufficient
for further studies. In the remaining 34 samples, morphological examination diagnosed
nine biopsies as Sertoli cell only (SCO, 26%), 12 as maturation arrest at spermatocyte
stage (MA), 12 as hypospermatogenesis with few sperm cells present (HS) and one sample
as obstructive azoospermia with normal spermatogenesis. A commercial total testicular
mRNA was used as a positive control for the gene expression.

Table 2 summarizes individual characteristics of the in vitro fertilization process, numbers of fertilized oocytes, embryo transfers and the cycles
as well as occurrence of clinical pregnancy. Fertilization rate for all studied samples
was 63%, in particular, for HS subset - 72% and MA subset – 54%. Pregnancy rate rate
was 29% for whole set of patients, from this 32% for HS group and 26% for MA group.
For samples with positive expression of studied genes fertilization rate for GAPDHS positive subset was 66%, ACR - 71%, SPATA22 – 68%, MND1 – 70%, pregnancy rates were 8%, 6%, 18% and 36% respectively.

Testicular biopsy of OA showed a similar expression pattern to that of commercial
testicular RNA (Figure 1). Three samples (10–12) from the HS group and six from the MA group (17–24) showed
no or low expression of the studied genes. In the SCO group, two samples (25 and 26
in Table 2) showed decreased expression of the tested genes, whereas in the remaining seven
biopsies only residual presence of GAPDHS, ACR and SPATA22 could be detected.

In patient 9 from the HS group and patients 22 and 23 from the MA group the expression
of MND1 and SPATA22 was detected and no ACR or GAPDHS gene products were found.

Next, we looked whether any difference in relative expression of the studied genes
could be found between the histological groups of HS, MA and SCO. Relative gene expression
was significantly decreased for SPATA22 and GAPDHS in the SCO group (Figure 2). The ACR gene was downregulated as well, but due to high inter-individual differences and
the low number of studied samples in the groups the decrease was not significant.

Figure 2.The relative difference in the expression of tested genes among control and experimental
groups of hypospermatogenesis (HS), maturation arrest (MA) and Sertoli cell- only
syndrome (SCO). Columns indicate the arithmetic mean of the relative expression of a particular gene,
whiskers indicate SEM. Control group (C) is indicated by purple color and the relative
expression was set as 100%. The statistically significant differences between control
and experimental groups are indicated by red color of the column or red asterisk.

Discussion

The main goal of all analytic procedures in patients with non-obstructive azoospermia
is to quickly obtain reliable data for successful prediction of testicular sperm retrieval.
Some laboratories attempted to predict spermatogenesis with non-invasive techniques
with differing success [14-16]. To date, the only generally accepted reliable predictor of successful TESE is testicular
histology [17]. Analysis of the germ cell-specific gene expression in testicular samples can provide
an additional, supplementing approach to increase the prediction of positive TESE
outcome.

Testicular transcriptome consists of gene expression patterns of both somatic and
germ cells and has been intensively studied in recent years [3]. The first studies were focused on describing the global testicular gene expression
and identifying testicular genes in mice [18] and human [19]. Shima et al. [1] took advantage of the first synchronous wave of spermatogenesis in pubertal mice
to locate the gene products to specific testicular cells. In a different approach,
germ cells were purified for high-throughput analysis of cell-specific gene expression
studies on animal models [20,21]. The data from the above-mentioned studies provides a vast number of possible gene
candidates as markers of spermatogenesis that fulfil the criteria of testis-specific
gene expression, are transcribed and translated at specific time points of spermatogenesis,
and their presence indicates correct gamete development. In addition, it was shown
recently that besides changes in mRNA levels in azoospermic men, the miRNA expression
is also altered [22].

Another approach to TESE sample analysis is to verify whether the expression of a
single gene or a couple of genes can be used as a simple indicator of positive sperm
retrieval in patients undergoing treatment in infertility clinics. Detection of DAZ
(deleted in azoospermia), DAZL (DAZ-like) and protamine 2 (PRM2) mRNA in testicular
samples was shown to be an informative tool for spermatogenesis evaluation [23]. Similar results were obtained with the BOULE mRNA occurrence [24]. Ando et al. showed that expression of VASA, ODF1, ODF2 (outer dense fiber 1 and 2) and SMCP (sperm mitochondria-associated cysteine-rich protein) genes was significantly stronger
in the successful TESE group [25]. Other genes expressed in post-meiotic stage may also be good candidates for the
prediction of successful fertilization.

In our study, we followed expression of five genes that are expressed at certain stages
of the spermatogenesis process and are important for meiosis and sperm development.
The MND1 and SPATA22 genes were selected for spermatogenesis characterization in azoospermic patients
because of the known significant gene expression differences in different non-obstructive
azoospermic patients [4]. The Mnd1/Gaj protein plays an important role in homologous chromosome pairing and
efficient cross-over during meiosis [26]. The SPATA22 gene product was shown to be involved in meiotic progression of germ cells in mice
[27]. The ACR mRNA appears first in pachytene spermatocytes, reaching the maximum levels in round
spermatids, and preproacrosin protein appears in spermatids [28]. The reason for non-obstructive azoospermia in this case may be interrupted or incomplete
spermiogenesis or sperm maturation. Nevertheless, the loss of acrosin protease activity
does not lead to infertility in mice and spermatozoa from knock-out mice can penetrate
zona pellucida of the oocyte [29]. The gene encoding sperm-specific glyceradehyde-3-phosphate dehydrogenase, GAPDHS, was shown to be expressed solely in haploid round and elongating spermatids [30,31] to replace the function of somatic GAPDH gene, whose expression ceases in germ cells. GAPDHS gene expression may be a good marker for spermatogenesis analysis, as its transcription
and translation are temporarily separated and mRNA forms a complex with an RNA-binding
protein, which results in translation and mRNA degradation delay [32]. Therefore, expression of the GAPDHS gene might be detectable even in poor-quality or low-quantity testicular samples.
Poor detection of gene expression in nine biopsies (10–12 from HS and 17–24 from MA
groups) suggests that in the tissue analysed for RNA purification, spermatogenesis
was either greatly reduced or RNA was probably purified mainly from somatic cells.
An interesting pattern of gene expression was observed in patients 9, 22 and 23 with
normal expression of MND1 and SPATA22 genes and residual levels of GAPDHS and ACR genes. This might indicate that spermatogenesis in these patients continues undisturbed
until meiosis, but either meiosis or spermiogenesis is somehow impaired.

Fertilization and pregnancy rates in population of studied patients were in accordance
to those from previous studies. Fertilization rates for subsets of samples with positive
expression of studied genes showed uniform fertilization rates around 70%, only MND1 gene was expressed in samples from SCO group where sperm cells could not be retrieved.
Surprisingly, for most promising markers of final steps of spermatogenesis, ACR and GAPDHS, pregnancy rate was below 10%. This indicates that expression analysis of present
testicular genes cannot indicate successful pregnancy in studied couples. It is highly
probable that in this process, oocyte and embryo quality have higher impact on the
successful pregnancy. Moreover, low number of studied samples does not allow drawing
any correlation between specific gene expression and fertilization outcome.

All four studied genes are expressed at different stages of spermatogenesis, and ACR, SPATA22 and GAPDHS gene expression might be a good predictor of successful TESE outcome. Nevertheless,
analysis of a greater number of testicular biopsies is needed to confirm that changes
in gene expression of the selected genes can serve as markers to justify repeated
TESE. Another thing to consider is that spermatogenesis is a dynamic process and TESE
sample analysis provides information about the gene expression and spermatogenesis
state at a single time point only.

A novel non-invasive approach to prediction of the state of spermatogenesis and pathophysiology
of testicular tissues via the detection of germ cell-specific mRNA traces in seminal
plasma was introduced recently [33,34]. Future analysis of germ cell-specific genes, including those from our study, or
GAPDH/GAPDHS ratio in cell-free seminal plasma from azoospermic patients might become a promising
non-invasive tool for TESE success prediction. The advantage of this technique is
that the seminal analysis provides complex whole-testis physiology in comparison to
the TESE sample representing a limited region of the analysed tissue.

To sum up, non-obstructive azoospermia is a complex pathophysiological state that
leads to changes of gene expression in the testes, and understanding this process
may lead to identification of the molecular markers of the spermatogenesis level.

Conclusions

Expression analysis of genes whose expression occurs exclusively in germ cells during
spermatogenesis provides sensitive confirmation of the histological diagnosis of SCO
syndrome, as it was decreased in all histologically identified SCO patients. In the
case of maturation arrest or hypospermatogenesis, gene expression analysis could help
determine the stage at which spermatogenesis arrest occurs and be a key factor in
making the decision whether repeated TESE could be considered after previous ICSI
failure.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

AD carried out the cDNA synthesis, qPCR analysis, participated in the study design
and drafted the manuscript; OT and KK collected the samples and corrected the manuscript;
EZ performed RNA work and critically read the manuscript; LD participated in the design
of the study and performed the statistical analysis; JP participated in the design
and coordination of the study and helped to draft the manuscript. All authors read
and approved the final manuscript.

Acknowledgements

This study was supported by grant No. P 503/1834 from the Grant Agency of the Czech
Republic and partly by the Institutional Research Support AV0Z 50520701.